Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2

J Biol Chem. 2001 Jun 29;276(26):23712-8. doi: 10.1074/jbc.M010002200. Epub 2001 Apr 24.


Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Binding Sites
  • Brain / enzymology*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Cattle
  • Cell Extracts / analysis
  • Chromatography, Gel
  • Cyclin-Dependent Kinase 5
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclin-Dependent Kinases / physiology*
  • Enzyme Activation
  • Glycogen Synthase Kinase 3
  • Glycogen Synthase Kinases
  • Macromolecular Substances
  • Magnesium / metabolism
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Phosphothreonine / metabolism
  • Protein Phosphatase 1
  • Proteins / metabolism*
  • Subcellular Fractions / enzymology


  • Cell Extracts
  • Macromolecular Substances
  • Proteins
  • protein phosphatase inhibitor-2
  • Phosphothreonine
  • Adenosine Triphosphate
  • Glycogen Synthase Kinases
  • Cyclin-Dependent Kinase 5
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Cyclin-Dependent Kinases
  • Glycogen Synthase Kinase 3
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1
  • Magnesium